The genus Haemophilus
contains a number of species of fastidious, gram-negative bacilli. Most of these are found as normal flora
of the upper respiratory tract. Haemophilus
species can cause infections in a variety of sites in the upper respiratory tract and elsewhere
in the body. Laboratory diagnosis is made by identifying these organisms in clinical specimens appropriately representing
the area of infection (throat swab, sinus drainage, sputum, conjunctival swab, spinal fluid, blood, or other). A direct smear of the
specimen may be useful, particularly for spinal fluid or an exudate from the eye, in providing rapid, presumptive information.
(Smears of material from the upper respiratory tract, with its mixed flora, may have little value unless the organisms are present
in large numbers.) Latex antibody tests can also be performed directly with certain patient body fluids to detect Haemophilus
antigen. Until an effective vaccine came into widespread use in the early 1990s, most serious Haemophilus
was caused by H. influenzae serogroup b (H. influenzae
strains are divided into serogroups a–f on the basis of their antigenic polysaccharide
capsule). This organism is seldom isolated in the clinical laboratory today, but other Haemophilus
species and H. influenzae
serogroups other than serogroup b are occasionally encountered.
The fastidious Haemophilus
organisms require specially enriched culture media and microaerophilic incubation conditions.
“Chocolate” agar is commonly used for primary isolation of Haemophilus
from clinical specimens. This medium contains
hemoglobin derived from bovine red blood cells as well as other enrichment growth factors. Because the hemoglobin is dark
brown, the agar in the plate has the appearance of chocolate.
Two special growth factors, called X and V, are required by some Haemophilus
species. Some require one but not the
other. The X factor is hemin
, a heat-stable derivative of hemoglobin (supplied in chocolate agar). The V factor is a heat-labile coenzyme
(nicotinamide adenine dinucleotide, or NAD), essential in the metabolism of some species that lack it. Yeast extracts contain
V factor and are one of the most convenient supplements of chocolate agar or other media used for Haemophilus
other than yeasts elaborate V factor. Staphylococci, for example, when growing on an agar plate secrete NAD into the surrounding
species that need V factor may grow in the zone immediately around the staphylococci but not elsewhere
on the plate. This growth of the dependent organism is described as “satellitism” (see colorplate 34
). X and V factors can also be
incorporated directly into agar media that do not contain these factors, or alternatively, they can be impregnated in filter-paper
disks that are pressed on the surface of X and V factor–deficient media. In the latter case, the growth factors diffuse into the agar
in a manner similar to diffusion from disks impregnated with antimicrobial agents (see Experiment 15.1).